- Even strains of the same species may exhibit differences in metabolite production, a finding that has important. - subtelomeres and uncover a polymorphic cluster present in only two strains that is closely related to the cluster responsible for biosynthesis of the mycotoxin aflatoxin (AF), a highly carcinogenic compound that is a major public health concern worldwide. - Full list of author information is available at the end of the article. - 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - Gene clusters involved in secondary metabolite (SM) production are exceptionally diverse and contain some of the most rapidly evolving gene families in fungi [1–3].. - These SMBGCs may contain one or multiple types of “core” enzymes responsible for bio- synthesis of the backbone structure of the metabolite.. - Analyzing SMBGCs across closely re- lated strains of the same species offers the opportunity to capture a snapshot of evolution and to better eluci- date the genome scale evolutionary processes shaping the birth and death of SMBGCs. - The ascomycete fungus Tolypocladium inflatum is an insect pathogen and ubiquitous soil saprotroph, best known as the producer of the lifesaving immunosuppressant drug. - The gen- ome of the isolate from which cyclosporin was originally isolated, T. - A previous karyo- type of the NRRL8044 strain of T. - However, each strain contained 6–7 large contigs greater than 1 Mb that aligned to the assembled chromosomes of the CBS714.70 strain (Additional file 2: Figure S1, Additional file 1: Table S1). - 75% of the total genome size (L75) was contained in the five largest. - Image of the globe is adapted from https://en.wikipedia.org/wiki/Template:Earth_Labelled_Map#/media/File:WorldMap.svg. - Telomeric repeats were also identified at both ends of a majority of the large contigs in strains NBRC31671, CBS567.84, and CBS824.70, and in over half of the larger contigs of NBRC31975 and NRRL8044 (Add- itional file 1: Table S1). - A heatmap of the pairwise inversion distance between strains closely followed the phylogeny based on SNPs, showing that strains CBS714.70 and NRRL8044 were separated by the most inversions, while CBS824.70 and CBS567.84 were most similar (Fig. - The end of chromo- some 2 in NRRL8044 was translocated to the end of the chromosome 6 (Fig. - In NRRL8044, a third large-scale intra-chromo- somal translocation involved movement of approximately 200 Kb from the right arm of chromosome 6 to the other arm of the same chromosome (Fig. - Phylogenetic analyses of the A domains of NRPSs, the ketosynthase (KS) domains of PKSs, and the TS do- mains of TSs was performed using maximum likelihood to identify homologous core genes (Additional file 10:. - III) An intrachromosomal inversion/translocation from the right arm of chromosome 6 to the opposite (left) arm of the same chromosome. - c Cartoon of each of the three translocation events. - These three gene clusters were part of the large 800 Kb translocation that moved them from the end of chromosome 2 to the end of chromosome 6 in NRRL8044 (Fig. - Of the 12 vari- able clusters that were not shared across all strains, 4 were shared by 5 strains (Fig. - Presence of telomeric repeats is indicated with a gray cap on the end of the chromosome. - chromosome) were moved as part of the large 800 Kb translocation of the end of chromosome 2 (orange) to the end of chromosome 6 (dark-blue) in 8044 (Fig. - Between panels B and C is a cartoon of the end of chromosome 6 in 8044, showing segments translocated from chromosome 2 (orange) and chromosome 4 (green). - Above this cartoon is a zoomed in genomic alignment of the region surrounding cluster E (red bar containing genes as yellow (T1-PKS) or blue (accessory gene) with b syntenic regions from chromosome 2 in all other strains (above, orange) showing that the T1-PKS and accessory genes are not found on chromosome 2 in other strains. - Below the cartoon is a genomic alignment of the translocated region surrounding cluster E on chromosome 6 in 8044 with (c) the region surrounding cluster 27 on chromosome 4 (below, green) in all other strains. - red shading), NRRL8044 contained a unique re- gion in the middle of the cluster consisting of a PKS gene and three additional accessory genes that showed homology to a portion of cluster 27 on chromosome 4 (Fig. - Genes flanking the 3′ end of the cluster were also highly conserved and syntenic (Fig. - Phylogenetic analyses of the KS domain showed that the core PKS gene in cluster E (Fig. - red shading), all other strains lacked the region containing the PKS and accessory genes in the middle of the cluster. - The movement of the PKS and three accessory genes from the middle of chromosome 4 to the end of. - Many of the variable clusters located within subtelomeric regions showed evidence of gain or loss of core genes and/or complete clusters. - In all strains except CBS714.70, Cluster 1, located on the left arm of chromosome 1, contained two large NRPSs com- prised of 13 and 8 modules, while the 13 modular gene and all but three modules of the 8 modular gene were deleted in CBS714.70. - Telomeric repeats were identi- fied on both arms of chromosome 1 in CBS714.70 and on the right arm of chromosome 1 in NRRL8044, suggesting these represent true losses rather than a failure to assem- ble these regions of the genome (Fig. - Blastn searches of the peptaibiotic NRPSs and DNA sequences from the cluster against the entire assem- bly also failed to recover any matching sequences.. - Alignment of the region surrounding cluster 1 across all strains showed that loss of the large NRPS core genes in CBS714.70 resulted from deletion of a ~ 88 Kb piece of DNA from the middle of the cluster (Fig. - The 3′ end of the cluster was. - conserved across strains and harbored all of the accessory genes found in other strains as well as a remnant pseudo- gene containing three NRPS A domains that grouped phylogenetically with the last three modules of the 8 modular NRPS in cluster 1 (Fig. - Alignment of the 5′ end of the cluster, however, showed that although one gene on the 5′ edge of the cluster was conserved across strains, the large 13 modular NRPS, as well as the first 5 modules of the 8 modular NRPS, were deleted in CBS714.70 (Fig. - found in all strains within the 5′ end of the cluster near the border of the region that was deleted in CBS714.70. - A LINE element was also found in the 5′ end of the cluster in strain NRRL8044 (Fig. - The entire 13 modular NRPS (containing 13 modules of A, T, and C domains numbered 1 – 13) and the first five modules of the 8 modular NRPS deleted in strain 714.70, leaving only a pseudogene comprised of 3 modules. - A DNA (hAT) TE (green) and a LINE/Tad1 retrotransposon (pink) are found at the edge of the deleted 13 modular NRPS within cluster 1 in strain 8044 and one DNA Mule (MuDr) element (green) is present on the 3 ′ end of the cluster in all T. - Homologs of the core PKS gene and nearly all add- itional accessory genes present in the T. - Phylogenetic analyses suggested that this relict A domain was most closely related to the first module of the 16 modular NRPS in T. - ophioglossoides and the first module of the 13 modular NRPS found in T. - arrows, Additional file 16: Figure S11), and showed that although there is not a one-to-one cor- respondence between A domains from different modules of the 16 modular NRPS in T. - The phylogenetic grouping of the relict A domain in cluster 42 on chromosome 6 with A domains from the 13 modular NRPS in cluster 1 on chromosome 1 in T. - An intriguing variable cluster, located in a subtelomeric region on the end of the left arm of chromosome 5 in only two strains (CBS824.70 and CBS567.84) (Fig. - Phylogenetic analyses of homologs of all genes in clus- ter D mined from a database of 340 other ascomycete taxa identified two distinct regions or subclusters of the complete cluster D that showed phylogenetic relation- ships consistent with independent co-diversification.. - Subcluster 1, spanning the 5′ end of the cluster, contained the four AF homologs (Fig. - genes Additional file 18: Figure S12) as well as additional accessory genes, and showed a distinct evolutionary history from genes located on the 3′ end of the cluster. - Similarly, phylogen- etic analyses of homologs of cluster genes mined from these 340 other ascomycete taxa identified a group of genes from the 3′ end of the cluster comprising subclus- ter 2 (Fig. - Two genes located in the middle of cluster (7744 and 7745), between the boundaries of the two subclusters, were conserved across all T. - Previous studies of diver- sity of the AF SMBGC across both Aspergillus species and isolates of A. - blue) contains a PKS (7740) and three additional genes with homology to aflC, aflT, aflA, and aflB, respectively, of the AF mycotoxin cluster of Aspergillus flavus. - c Mauve alignment of the region of the chromosome containing cluster D and the syntenic regions in other strains revealed numerous genome rearrangements within and surrounding cluster D. - same four genes occur within one of the largest linkage disequilibrium (LD) blocks (Fig. - genes Additional file 19: Table S7) of the cluster. - Interestingly, none of the genes from cluster D were present in strain NRRL8044 (Fig. - Strain NBRC31975 was the only strain that contained genes from both subcluster 1 and subcluster 2 as well as the two genes in the middle of the cluster (7744 and 7745)(Fig. - Genomic alignments of the region surrounding the cluster across all T. - of the cluster (Fig. - Conservation of the Cyclosporin biosynthetic cluster In contrast to these rapidly evolving clusters located on the ends of chromosomes, several clusters located more internally on chromosomes, including the cyclosporin cluster, were highly conserved. - Genomic alignments showed synteny and complete con- servation of gene content of the cyclosporin gene cluster across all T. - Analyses of selection of the cyclosporin synthase gene (simA), responsible for biosynthesis of the peptide backbone structure of cyclo- sporin, found no codon positions under positive selection and showed an average dN/dS ratio of 0.204 (Additional file 20: Table S8). - Several other clusters located internally on chromosomes, including a cluster containing homologs of the fumonisin biosynthetic cluster in F. - 9 Conservation of the CsA cluster. - Mauve alignment of the SMBGC responsible for CsA biosynthesis (cluster 26) in the middle of chromosome 4 across all six T. - SMBGC through a series of translocation events that bring together different parts of the cluster from three different chromosomes. - In other organ- isms, ranging from humans to yeast to the malarial para- site Plasmodium falciparum, subtelomeres are highly dynamic regions of the genome [56, 57], comprising hot- spots for recombination between both homologous and heterologous chromosomes [58, 59], as well as for inser- tion and deletion events [57]. - As core SM genes are essential for production of the backbone structure of SMs, their gain, loss, or move- ment within the genome has potential to generate dra- matic changes in metabolites produced. - In addition to LTR transposons observed in close vicinity to several of the variable clusters in T. - The DNA-hAT trans- poson Restless was previously found in the 5′ flanking re- gion of a cluster involved in the production of destruxins, another class of immune modulating peptides produced by the related insect pathogenic fungus Metarhizium [65], leading to degeneration of the cluster and inability to pro- duce destruxins. - Modular evolution of a polymorphic AF-like cluster The large polymorphic cluster D in the subtelomeric re- gion of the left arm of chromosome 5 is a composite cluster composed of at least two independently co-diversifying subclusters, each containing genes that. - AFs are carcinogenic polyketides that are produced primarily by Aspergillus species [66] and AF B1 is considered one of the most toxigenic substances known. - Other clusters containing homologs of genes in the AF cluster include the cluster responsible for biosynthesis of the toxic metabolite DOTH in the pine pathogen Dothis- troma septosporum, and related clusters in other asco- mycetes that contain at least three of the four AF homologs (aflC, aflB, and aflA) [21] (Fig. - In our study, the gene trees of the four homologs of AF genes in T.. - A previous study of dermatophyte fungi identi- fied two distinct SMBGCs occupying the same syntenic location and concluded that an ancestral cluster contain- ing all elements found in the locus underwent differen- tial loss of parts of the cluster in different species, leading to distinct cluster types in extant species [26].. - inflatum, may be a more likely explanation of the discontinuous distribution of this cluster within T. - The cluster is located in a subtelomeric region showing evidence of multiple inver- sions and other rearrangements and several strains con- tain multiple TEs on either side of the cluster. - Chromosome scale assemblies of six strains of the cyclo- sporin producing fungus T. - inflatum enabled examin- ation of the role of genome architecture and. - Our re- sults demonstrate that movement of core SM genes (PKS, NRPS, TS, DMAT) involved in production of the metabolite backbone structures may occur more fre- quently in fungal genomes than previously recognized and contributes substantially to the evolution of novel metabolite clusters in fungi. - 50 kb to any of the major contigs. - A codon alignment for each of four partitions of the simA gene were created using pal2nal [91]. - 18184432) to construct an alignment graph that guides the final assembly of the multi-genome alignment. - Blast hits of the Cluster D genes to A.. - L75: >75% of the total genome size. - The funding agency had no role in in the design of the study, collection, analysis, and interpretation of data, or in writing the manuscript.. - Gene duplication, modularity and adaptation in the evolution of the aflatoxin gene cluster.. - Deciphering the cryptic genome: genome-wide analyses of the rice pathogen Fusarium fujikuroi reveal complex regulation of secondary metabolism and novel metabolites. - The genome of the truffle-parasite Tolypocladium ophioglossoides and the evolution of antifungal peptaibiotics.. - Two different secondary metabolism gene clusters occupied the same ancestral locus in fungal dermatophytes of the Arthrodermataceae. - Plant-symbiotic Fungi as chemical engineers: multi-genome analysis of the Clavicipitaceae reveals dynamics of alkaloid loci. - The genome of Tolypocladium inflatum: evolution, organization and expression of the cyclosporin biosynthetic gene cluster. - Analysis of the Trichoderma virens NRPS gene, tex1: expression and effect on biocontrol. - Genomic sequence of the pathogenic and allergenic filamentous fungus aspergillus fumigatus. - Multiple translocation of the AVR-Pita effector gene among chromosomes of the rice blast fungus Magnaporthe oryzae and related species
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